Justin Yan's CIAP Aptamer Project (2016)

The CIAP-Aptamer Complex: A Potential Source of One Agent Detection

Introduction/Background

When it comes to detection systems, ELISA is one of the most reliable tests used to detect the presence of a number of diseases including HIV, Lyme disease, syphilis, and more diseases based off of particular antibody presence in blood. However, the necessary materials don’t come cheap; an insulin kit with 96 assays can cost up to 500 dollars per kit. As a result, we are looking to develop an alternate aptamer-based version (ELONA) of the ELISA to reduce costs and increase binding efficiency.

CIAP, also known as Calf Intestinal Alkaline Phosphatase, is an alkaline phosphatase involved in the removal of phosphates from 5’ ends of nucleic acids to prevent ligation. This 69 kDa protein has an isoelectric point of 5.7, is partially negatively charged, and can be stored in a basic buffer due to the nature of its phosphate removal function. Normally found as a dimer, it is commonly used for removal of phosphate groups for radiolabelling, enzyme immunoassay labeling, and detection of undifferentiated pluripotent stem cells. The one particular use in which this study focuses upon is CIAP’s role as a reporter molecule. After antibodies are applied to the surface such that they can bind to the antigen specifically, CIAP bound to the antibody can phosphorylate the substrate PNPP to generate a color change; fluorimetry can then be performed to identify the protein complex presence and the amount of complex in a medium via measurements of fluorescence.

However, antibodies, which are used in the formation of the generalized CIAP-antibody complex, have reduced immunoreactivity in ELISA kits. By replacing antibodies with aptamer, this would greatly assist in the development of a cheaper and more reliable one-agent detection system (Huynh 2015).

Aptamers are random sequence single stranded RNA or DNA nucleic acid strands that are produced cheaply in vitro in a SELEX bead-based process; these molecules can bind to pre-selected targets such as proteins, nucleic acids, and small organic compounds with high affinity and specificity (Figure 2). The most common sources of aptamers are riboswitches, segments of an mRNA that can bind to a small target molecule, concentrations of which directly regulate the riboswitch’s activity (Kumar 2015). Aptamers involving CIAP specifically have been used in institutions such as McMasters University and the University of Texas at Austin, and have already been developed to treat age related macular degeneration (Macugen) and act as bioimaging probes. In this scenario, however, this aptamer will serve as a diagnostic tool via antibody replacement in ELISA detection systems.

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References